Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on three groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder hereditary inclusion body myopathy (HIBM). In 2007, we characterized a knock-in HIBM mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice and is a promising treatment for human patients (J Clin Inv (2007) 117:1585-1594). Our ManNAc patent (No. 60/932,451) was licensed to a ManNAc manufacturer, New Zealand Pharmaceuticals. And from 2010-2012 preclinical studies have been performed with assistance from the NIH-TRND (Therapies for Rare and Neglected Diseases) program, leading to an IND (Investigational New Drug) application and approval from FDA for the use of ManNAc in HIBM patients. In 2011 a natural history study (11-HG-0218, NHGRI) of patients with Hereditary Inclusion Body Myopathy (HIBM) was initiated, and in 2012 a Phase 1 study started (T-HG-0082, NHGRI): A Phase 1 Randomized, Escalating Single-Dose Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of ManNAc in Subjects with Hereditary Inclusion Body Myopathy (HIBM), for both clinical studies, our group coordinates/performs sample collection, storage and research. In the last year, we wrote an extensive review on all aspects of GNE (Current Biology, under review), identified novel GNE isoforms in human and mice and performed molecular modeling on these isoforms (Ref. 1), performed biochemical analysis for a gene therapy trial of a human HIBM patient (Ref. 2), optimized a retro-orbital injection method for compounds into newborn mice (Ref. 3), defined the kidney phenotype in our HIBM mouse model and used this model to develop a lectin panel for screening human kidney disorders for hyposialylation (Ref.4), and evaluated the use of other sugars in the sialic acid synthesis pathway as alternative therapeutic options to increase sialylation (Mol Genet Metab, under review). We tested the lectin panel, developed with use of our HIBM mouse model (Ref.4), on a variety of unexplained human renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. Human renal disorders involving glomerular hyposialylation may benefit from ManNAc as a therapeutic agent (J Exp Med, under review). This last year we also further characterized the adult onset muscle phenotype of our HIBM knock-in mouse model and tested alternative HIBM treatments on our murine model, including feeding with sialic acid pathway intermediates and GNE gene therapy, mostly intravenous delivered embedded in liposomes (Lipoplex). The results are being compiled for a manuscript. We acquired a provisional patent for the use of liposomes to systemically deliver saccharides (i.e., ManNAc and sialic acid) to mammals (Ref. 10). Our further studies focused on sub-cellular localization and expression levels of GNE and other enzymes in the sialic acid synthesis pathway (Western blotting and real-time quantitative PCR) and identifiying human biomarkers that can serve as parameters for sialylation status (mainly by glycan-profiling studies). 2. Disorders of lysosome-related organelles (LRO) biogenesis. Such disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, Gray Platelet syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. We investigate known and unknown LRO-disorders-causing genes (by conventional and next generation sequencing techniques), with the goal of better understanding the biology of the disease. To study the effects of LRO-disorders mutations, we perform cell biological studies on patient material (using immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in cells. Such cells fail to transport certain lysosome-related organelle resident proteins to their correct destinations, and LRO-disorder gene products are generally involved in recognizing the specific vesicles that give rise to LROs. We also catalogue the clinical and genetic characteristics of the distinct subtypes of HPS and related LRO-disorders. This last year we identified NBEAL2 as the long sought-after gene causing Gray Platelet syndrome (Ref.5), established that pulmonary fibrosis is associated with HPS type 2 (Ref.6), assisted in identification of involvement of the BLOS1-Interacting Protein KXD1 in the Biogenesis of Lysosome-Related Organelles (Ref.7), characterized Griscelli syndrome type 3 cases (Ref.8). 3. Genetics of Smith-Magenis syndrome (SMS) and related disorders. SMS is a complex neurobehavioral disorder characterized by multiple congenital anomalies, primarily ascribed to a de novo interstitial deletion of 17p11.2. Molecular analysis of SMS patients may shed light on the variable phenotype and genotype-phenotype correlations and possible treatment decisions. The NIH cohort of SMS patients contains patients with the common 3.7 Mb 17p11.2 deletion (n=80), with atypical 17p11.2 deletions (n=24), and non 17p11.2 deleted patients (n= 44). Our whole genome SNP-array analysis on the atypical deletion group identified different 17p11.2 breakpoints that may influence clinical features (manuscript in preparation). SNP-array analysis of the non-deleted cohort identified several novel (micro) deletions or duplications. Our extensive RAI1 gene analysis on the non-deleted subgroup, including mutation analysis and expression studies, identified 10 patients with RAI1 mutations (5 de novo, 5 familial) and described for the first time decreased RAI1 mRNA expression levels not only in patients with the common 17p11.2 deletion but also in RAI1 mutated patients, and in some non-17p11.2 deleted patients (Ref. 9).